SHP-2 regulates the phosphatidylinositide 3'-kinase/Akt pathway and suppresses caspase 3-mediated apoptosis

The Src homology domain 2 (SH2)-containing tyrosine phosphatase SHP-2 has been implicated in the regulation of the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway. The ability of SHP-2 to regulate the PI3K/Akt pathway is suggested to result in the positive effect of SHP-2 on cell survival. Whether SHP-2 regulates insulin-like growth factor-1 (IGF-1)-dependent activation of Akt at the level of PI3K has yet to be established. Furthermore, the identification of the down-stream apoptotic target engaged by SHP-2 in cell survival also has yet to be determined. Here, we show that overexpression of a catalytically inactive mutant of SHP-2 inhibited insulin-like growth factor-1 (IGF-1)-dependent PI3K and Akt activation. Consistent with the observation that SHP-2 participates in pro-survival signaling fibroblasts expressing a deletion within exon 3 of SHP-2, which results in a truncation of the amino-terminus SH2 domain (SHP-2(Ex3-/-)), were hypersensitive to etoposide-induced cell death. SHP-2(Ex3-/-) fibroblasts exhibited enhanced levels of etoposide-induced caspase 3 activity as compared to wild-type fibroblasts and the enhanced level of caspase 3 activity was suppressed by a caspase 3-specific inhibitor. Re-introduction of wild-type SHP-2 into the SHP-2(Ex3-/-) fibroblasts rescued the hypersensitivity to etoposide-induced caspase 3 activation. The effects of abrogating SHP-2 function on cell survival were not specific to the loss of the amino-terminus SH2 domain of SHP-2 since RNAi-mediated knock-down of SHP-2 also reduced cell survival. Taken together, these data indicate that the catalytic activity of SHP-2 is required to regulate the PI3K/Akt pathway and thus likely participates in anti-apoptotic signaling by suppressing caspase 3-mediated apoptosis.     

Functional analysis of aortic endothelial cells expressing mutant PDGF receptors with respect to expression of matrix metalloproteinase-3

Platelet-derived growth factor (PDGF) stimulates expression of matrix metalloproteinases (MMPs), including stromelysin-1 (MMP-3). Induction of these expressions is known to occur during the course of atherosclerosis, tumor invasion, and metastasis. We investigated PDGF-alpha receptor (alphaR)- and beta receptor (betaR)-mediated signaling pathways for the expression of MMP-3 and invasion activity using porcine aortic endothelial (PAE) cells with stable expression of normal or mutated PDGF receptors. RT-PCR and Western blot analyses revealed that PDGF-BB induces MMP-3 expression in PAE cells that exclusively express either the PDGF-alphaR or the -betaR, but not in non-transfected control cells. To identify the signals necessary for PDGF receptor-mediated induction of MMP-3 expression, several lines of PAE cells expressing mutant PDGF receptors were further analyzed. Cells expressing mutant PDGF receptors unable to associate with Src or PLCgamma, retained the ability to induce MMP-3 expression as a result of PDGF-BB stimulation. However, incubation with PDGF-BB did not induce MMP-3 expression in cells expressing a mutant PDGF-betaR unable to associate with phosphatidylinositol 3(')-kinase (PI3K). LY294002, a PI3K inhibitor, reduced PDGF-BB-stimulated MMP-3 expression in PAE cells expressing wild-type PDGF receptors. In contrast, PDGF-BB induced MMP-3 expression in the presence of U-73122, a PLCgamma inhibitor. Moreover, PDGF-BB enhanced the invasiveness of cells expressing wild type PDGF-beta receptors, but not of cells expressing mutant PDGF-betaRs impaired in their ability to associate with PI3K. In light of these results, it appears that PDGF-BB is capable of inducing MMP-3 expression through both the PDGF-alphaR and the -betaR, and the effects are contributed by the PI3K-mediated transduction pathways.     

Signal transduction pathways triggered by fibroblast growth factor receptor 1 expressed in Xenopus laevis oocytes after fibroblast growth factor 1 addition. Role of Grb2, phosphatidylinositol 3-kinase, Src tyrosine kinase, and phospholipase Cgamma.

Xenopus oocytes expressing fibroblast growth factor receptor 1 (FGFR1) were used as a biological model system to analyse the signal transduction pathways that are triggered by fibroblast growth factor 1 (FGF1). Germinal vesicle breakdown (GVBD) and phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2) occured 15 h after FGF1 addition. These events were Ras-dependent as they were blocked by a Ras dominant negative form. The Ras activity was promoted by three upstream effectors, growth factor-bound protein 2 (Grb2), phosphatidylinositol 3-kinase (PI3K) and Src cytoplasmic kinase. Ras activation was inhibited by a Grb2 dominant negative form (P49L), by PI3K inhibitors, including wortmannin, LY294002, the N-SH2 domain of p85alpha PI3K and by the SH2 domain of Src. Src activation induced by FGF1 was blocked by the SH2 domain of Src and PP2, a specific inhibitor of Src. The Grb2 adaptor was recruited by the upstream Src homology 2/alpha-collagen-related (Shc) effector, as the SH2-Shc domain prevented the GVBD and the ERK2 phosphorylation induced by FGF1. The importance of another signalling pathway involving phospholipase Cgamma (PLCgamma) was also investigated. The use of the PLCgamma inhibitory peptide, neomycin and the calcium chelator BAPTA-AM on oocytes expressing FGFR1 or the stimulation by PDGF-BB of oocytes expressing PDGFR-FGFR1 mutated on the PLCgamma binding site, prevented GVBD and ERK2 phosphorylation. This study shows that the transduction cascade induced by the FGFR1-FGF1 interaction in Xenopus oocytes represents the sum of Ras-dependent and PLCgamma-dependent pathways. It emphasizes the role played by PI3K and Src and their connections with the Ras cascade in the FGFR1 signal transduction.     

Phosphatidylinositol 3-kinase-dependent membrane recruitment of Rac-1 and p47phox is critical for alpha-platelet-derived growth factor receptor-induced production of reactive oxygen species

Platelet-derived growth factor (PDGF) plays a critical role in the pathogenesis of proliferative diseases. NAD(P)H oxidase (Nox)-derived reactive oxygen species (ROS) are essential for signal transduction by growth factor receptors. Here we investigated the dependence of PDGF-AA-induced ROS production on the cytosolic Nox subunits Rac-1 and p47(phox), and we systematically evaluated the signal relay mechanisms by which the alphaPDGF receptor (alphaPDGFR) induces ROS liberation. Stimulation of the alphaPDGFR led to a time-dependent increase of intracellular ROS levels in fibroblasts. Pharmacological inhibitor experiments and enzyme activity assays disclosed Nox as the source of ROS. alphaPDGFR activation is rapidly followed by the translocation of p47(phox) and Rac-1 from the cytosol to the cell membrane. Experiments performed in p47(phox)(-/-) cells and inhibition of Rac-1 or overexpression of dominant-negative Rac revealed that these Nox subunits are required for PDGF-dependent Nox activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA-dependent ROS production, we investigated Ph cells expressing mutant alphaPDGFRs that lack specific binding sites for alphaPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase (PI3K), phospholipase Cgamma, and SHP-2). Lack of PI3K signaling (but not Src, phospholipase Cgamma, or SHP-2) completely abolished PDGF-dependent p47(phox) and Rac-1 translocation, increase of Nox activity, and ROS production. Conversely, a mutant alphaPDGFR able to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110alpha (but not p110beta) was identified as the crucial isoform that elicits alphaPDGFR-mediated production of ROS. Finally, bromodeoxyuridine incorporation and chemotaxis assays revealed that the lack of ROS liberation blunted PDGF-AA-dependent chemotaxis but not cell cycle progression. We conclude that PI3K/p110alpha mediates growth factor-dependent ROS production by recruiting p47(phox) and Rac-1 to the cell membrane, thereby assembling the active Nox complex. ROS are required for PDGF-AA-dependent chemotaxis but not proliferation.     

Identification of the receptor-associated signaling enzymes that are required for platelet-derived growth factor-AA-dependent chemotaxis and DNA synthesis.

Activation of the platelet-derived growth factor (PDGF) alpha receptor (alphaPDGFR) leads to cell migration and DNA synthesis. These events are preceded by the ligand-induced tyrosine phosphorylation of the receptor and its association with SH2-containing signaling enzymes including Src family members (Src), the phosphotyrosine phosphatase SHP-2, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-gamma1 (PLCgamma). In this study, we sought to systematically evaluate the relative roles of the signaling enzymes that are recruited to the alphaPDGFR for DNA synthesis and cell migration. Our approach was to generate and characterize tyrosine to phenylalanine alphaPDGFR mutants that failed to associate with one or more of the above listed signaling enzymes. In a 3T3-like cell line (Ph cells), PDGF-dependent DNA synthesis was strictly dependent on only one of the receptor-associated proteins, PI3K. In contrast, multiple signaling enzymes were required for maximal chemotaxis, as receptors unable to associate with either Src, PI3K, or PLCgamma initiated chemotaxis to 4, 47, or 56% of the wild-type level, respectively. Furthermore, coexpression of mutant receptors revealed that these signaling enzymes do not need to be on the same receptor for a cell to respond chemotactically to PDGF. We conclude that for the alphaPDGFR, PI3K plays a major role in initiating DNA synthesis, whereas PI3K, PLCgamma, and especially Src are required for chemotaxis.     

Signaling complexes and protein-protein interactions involved in the activation of the Ras and phosphatidylinositol 3-kinase pathways by the c-Ret receptor tyrosine kinase

Proximal signaling events and protein-protein interactions initiated after activation of the c-Ret receptor tyrosine kinase by its ligand, glial cell line-derived neurotrophic factor (GDNF), were investigated in cells carrying native and mutated forms of this receptor. Mutation of Tyr-1062 (Y1062F) in the cytoplasmic tail of c-Ret abolished receptor binding and phosphorylation of the adaptor Shc and eliminated activation of Ras by GDNF. Phosphorylation of Erk kinases was also greatly attenuated but not eliminated by this mutation. This residual wave of Erk phosphorylation was independent of the kinase activity of c-Ret. Mutation of Tyr-1096 (Y1096F), a binding site for the adaptor Grb2, had no effect on Erk activation by GDNF. Activation of phosphatidylinositol-3 kinase (PI3K) and its downstream effector Akt was also reduced in the Y1062F mutant but not completely abolished unless Tyr-1096 was also mutated. Ligand stimulation of neuronal cells induced the assembly of a large protein complex containing c-Ret, Grb2, and tyrosine-phosphorylated forms of Shc, p85(PI3K), the adaptor Gab2, and the protein-tyrosine phosphatase SHP-2. In agreement with Ras-independent activation of PI3K by GDNF in neuronal cells, survival of sympathetic neurons induced by GDNF was dependent on PI3K but was not affected by microinjection of blocking anti-Ras antibodies, which did compromise neuronal survival by nerve growth factor, suggesting that Ras is not required for GDNF-induced survival of sympathetic neurons. These results indicate that upon ligand stimulation, at least two distinct protein complexes assemble on phosphorylated Tyr-1062 of c-Ret via Shc, one leading to activation of the Ras/Erk pathway through recruitment of Grb2/Sos and another to the PI3K/Akt pathway through recruitment of Grb2/Gab2 followed by p85(PI3K) and SHP-2. This latter complex can also assemble directly onto phosphorylated Tyr-1096, offering an alternative route to PI3K activation by GDNF.     

The scaffolding adapter Gab1 mediates vascular endothelial growth factor signaling and is required for endothelial cell migration and capillary formation

Vascular endothelial growth factor (VEGF) is involved in the promotion of endothelial cell proliferation, migration, and capillary formation. These activities are mainly mediated by the VEGFR2 receptor tyrosine kinase that upon stimulation, promotes the activation of numerous proteins including phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase (PI3K), Akt, Src, and ERK1/2. However, the VEGFR2-proximal signaling events leading to the activation of these targets remain ill defined. We have identified the Gab1 adapter as a novel tyrosine-phosphorylated protein in VEGF-stimulated cells. In bovine aortic endothelial cells, Gab1 associates with VEGFR2, Grb2, PI3K, SHP2, Shc, and PLCgamma, and its overexpression enhances VEGF-dependent cell migration. Importantly, silencing of Gab1 using small interfering RNAs leads to the impaired activation of PLCgamma, ERK1/2, Src, and Akt; blocks VEGF-induced endothelial cell migration; and perturbs actin reorganization and capillary formation. In addition, co-expression of VEGFR2 with Gab1 mutants unable to bind SHP2 or PI3K in human embryonic kidney 293 cells and bovine aortic endothelial cells mimics the defects observed in Gab1-depleted cells. Our work thus identifies Gab1 as a novel critical regulatory component of endothelial cell migration and capillary formation and reveals its key role in the activation of VEGF-evoked signaling pathways required for angiogenesis.     

Reactive oxygen species regulate M-CSF-induced monocyte/macrophage proliferation through SHP1 oxidation

Macrophage colony-stimulating factor (M-CSF) stimulation results in the production of reactive oxygen species (ROS) that participate in the proliferation of monocyte/macrophage. However, the molecular mechanisms whereby ROS modulate the signaling processes of M-CSF remain poorly defined. We report here that the redox-sensitive Src homology region 2 domain-containing phosphatase 1 (SHP1) is a critical regulator of M-CSF-mediated signaling in bone marrow monocyte/macrophage lineage cells (BMMs). Application of diphenylene iodonium (DPI) inhibited the responses of BMMs to M-CSF, including ROS production, cell proliferation, and phosphorylation of c-Fms as well as Akt kinase, but not of MAP kinases such as ERK, p38, and JNK. Dysregulation of SHP1 by overexpression or RNA interference in BMMs showed that SHP1 specifically regulates PI3 kinase (PI3K)/Akt signaling, but not MAP kinases in a redox-dependent manner, thereby regulating proliferation of BMMs through cyclins D1 and D2. These findings demonstrate that M-CSF-mediated ROS generation leads to SHP1 oxidation, which promotes cell proliferation through the PI3K/Akt-dependent signaling pathway.     

SHP-1 regulates Lck-induced phosphatidylinositol 3-kinase phosphorylation and activity.

Ligation of the T cell antigen receptor (TCR) activates the Src family tyrosine kinase p56 Lck, which, in turn, phosphorylates a variety of intracellular substrates. The phosphatidylinositol 3-kinase (PI3K) and the tyrosine phosphatase SHP-1 are two Lck substrates that have been implicated in TCR signaling. In this study, we demonstrate that SHP-1 co-immunoprecipitates with the p85 regulatory subunit of PI3K in Jurkat T cells, and that this association is increased by ligation of the TCR complex. Co-expression of SHP-1 and PI3K with a constitutively activated form of Lck in COS7 cells demonstrated the carboxyl-terminal SH2 domain of PI3K to inducibly associate with the full-length SHP-1 protein. By contrast, a truncated SHP-1 mutant lacking the Lck phosphorylation site (Tyr(564)) failed to bind p85. Wild-type but not catalytically inactive SHP-1 induced dephosphorylation of p85. Furthermore, expression of SHP-1 decreased PI3K enzyme activity in anti-phosphotyrosine immunoprecipitates and phosphorylation of serine 473 in Akt, a process dependent on PI3K activity. These results indicate the presence of a functional interaction between PI3K and SHP-1 and suggest that PI3K signaling, which has been implicated in cell proliferation, apoptosis, cytoskeletal reorganization, and many other biological activities, can be regulated by SHP-1 in T lymphocytes.     

Src homology domain 2-containing protein-tyrosine phosphatase-1 (SHP-1) binds and dephosphorylates G(alpha)-interacting, vesicle-associated protein (GIV)/Girdin and attenuates the GIV-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway

GIV (Gα-interacting vesicle-associated protein, also known as Girdin) is a bona fide enhancer of PI3K-Akt signals during a diverse set of biological processes, e.g. wound healing, macrophage chemotaxis, tumor angiogenesis, and cancer invasion/metastasis. We recently demonstrated that tyrosine phosphorylation of GIV by receptor and non-receptor-tyrosine kinases is a key step that is required for GIV to directly bind and enhance PI3K activity. Here we report the discovery that Src homology 2-containing phosphatase-1 (SHP-1) is the major protein-tyrosine phosphatase that targets two critical phosphotyrosines within GIV and antagonizes phospho-GIV-dependent PI3K enhancement in mammalian cells. Using phosphorylation-dephosphorylation assays, we demonstrate that SHP-1 is the major and specific protein-tyrosine phosphatase that catalyzes the dephosphorylation of tyrosine-phosphorylated GIV in vitro and inhibits ligand-dependent tyrosine phosphorylation of GIV downstream of both growth factor receptors and GPCRs in cells. In vitro binding and co-immunoprecipitation assays demonstrate that SHP-1 and GIV interact directly and constitutively and that this interaction occurs between the SH2 domain of SHP-1 and the C terminus of GIV. Overexpression of SHP-1 inhibits tyrosine phosphorylation of GIV and formation of phospho-GIV-PI3K complexes, and specifically suppresses GIV-dependent activation of Akt. Consistently, depletion of SHP-1 enhances peak tyrosine phosphorylation of GIV, which coincides with an increase in peak Akt activity. We conclude that SHP-1 antagonizes the action of receptor and non-receptor-tyrosine kinases on GIV and down-regulates the phospho-GIV-PI3K-Akt axis of signaling.     

Phosphatidylinositol 3-kinase signaling is involved in neurogenesis during Xenopus embryonic development

Phosphatidylinositol 3-kinase (PI3K) has numerous cellular functions, including cell survival and proliferation. In this study, we demonstrated that the expression of the active form of PI3K induced dorsal differentiation and axis duplication and strongly induced the expression of neural markers. In contrast, the inhibition of PI3K activity by its dominant negative mutant induced the phenotype of losing posterior structures and the expression of ventral markers. Akt is an essential target of PI3K for neurogenesis. The expression of the active form of Akt induced axis duplication and increased the expression of neural markers. Inhibition of the Akt activity abolished the PI3K-induced double heads and axes. This signal transmits through its target, glycogen synthase kinase 3beta, which is known to mediate Wnt signaling for Xenopus development. These results identify a new function of PI3K/Akt signaling in axis formation and neurogenesis during Xenopus embryonic development and provide a direct link between growth factor-mediated PI3K/Akt signaling and Wnt signaling during embryonic development.     

Pathway crosstalk between Ras/Raf and PI3K in promotion of M-CSF-induced MEK/ERK-mediated osteoclast survival

While M-CSF-mediated MEK/ERK activation promotes osteoclast survival, the signaling pathway by which M-CSF activates MEK/ERK is unresolved. Functions for PI3K, Ras, and Raf have been implicated in support of osteoclast survival, although interaction between these signaling components has not been examined. Therefore, the interplay between PI3K, Ras and Raf in M-CSF-promoted MEK/ERK activation and osteoclast survival was investigated. M-CSF activates Ras to coordinate activation of PI3K and Raf/MEK/ERK, since Ras inhibition decreased PI3K activation and PI3K inhibition did not block M-CSF-mediated Ras activation. As further support for Ras-mediated signaling, constitutively active (ca) Ras promoted MEK/ERK activation and osteoclast survival, which was blocked by inhibition of PI3K or Raf. Moreover, PI3K-selective or Raf-selective caRas were only partially able to promote osteoclast survival when compared to parental caRas. We then examined whether PI3K and Raf function linearly or in parallel downstream of Ras. Expression of caPI3K increased MEK/ERK activation and promoted osteoclast survival downstream of M-CSF, supporting this hypothesis. Blocking Raf did not decrease osteoclast survival and MEK/ERK activation promoted by caPI3K. In addition, PI3K-selective Ras-mediated survival was not blocked by Raf inhibition. Taken together, our data support that Raf signaling is separate from Ras/PI3K signaling and PI3K signaling is separate from Ras/Raf signaling. These data therefore support a role for Ras in coordinate activation of PI3K and Raf acting in parallel to mediate MEK/ERK-promoted osteoclast survival induced by M-CSF.     

Src Kinase and Syk Activation Initiate PI3K Signaling by a Chimeric Latent Membrane Protein 1 in Epstein-Barr Virus (EBV)+ B Cell Lymphomas

The B lymphotrophic γ-herpesvirus EBV is associated with a variety of lymphoid- and epithelial-derived malignancies, including B cell lymphomas in immunocompromised and immunosuppressed individuals. The primary oncogene of EBV, latent membrane protein 1 (LMP1), activates the PI3K/Akt pathway to induce the autocrine growth factor, IL-10, in EBV-infected B cells, but the mechanisms underlying PI3K activation remain incompletely understood. Using small molecule inhibition and siRNA strategies in human B cell lines expressing a chimeric, signaling-inducible LMP1 protein, nerve growth factor receptor (NGFR)-LMP1, we show that NGFR-LMP1 utilizes Syk to activate PI3K/Akt signaling and induce IL-10 production. NGFR-LMP1 signaling induces phosphorylation of BLNK, a marker of Syk activation. Whereas Src kinases are often required for Syk activation, we show here that PI3K/Akt activation and autocrine IL-10 production by NGFR-LMP1 involves the Src family kinase Fyn. Finally, we demonstrate that NGFR-LMP1 induces phosphorylation of c-Cbl in a Syk- and Fyn-dependent fashion. Our results indicate that the EBV protein LMP1, which lacks the canonical ITAM required for Syk activation, can nevertheless activate Syk, and the Src kinase Fyn, resulting in downstream c-Cbl and PI3K/Akt activation. Fyn, Syk, and PI3K/Akt antagonists thus may present potential new therapeutic strategies that target the oncogene LMP1 for treatment of EBV+ B cell lymphomas.     

Intestinal epithelial cancer cell anoikis resistance: EGFR‐mediated sustained activation of Src overrides Fak‐dependent signaling to MEK/Erk and/or PI3‐K/Akt‐1

Herein, we investigated the survival roles of Fak, Src, MEK/Erk, and PI3‐K/Akt‐1 in intestinal epithelial cancer cells (HCT116, HT29, and T84), in comparison to undifferentiated and differentiated intestinal epithelial cells (IECs). We report that: (1) cancer cells display striking anoikis resistance, as opposed to undifferentiated/differentiated IECs; (2) under anoikis conditions and consequent Fak down‐activation, cancer cells nevertheless exhibit sustained Fak–Src interactions and Src/MEK/Erk activation, unlike undifferentiated/differentiated IECs; however, HCT116 and HT29 cells exhibit a PI3‐K/Akt‐1 down‐activation, as undifferentiated/differentiated IECs, whereas T84 cells do not; (3) cancer cells require MEK/Erk for survival, as differentiated (but not undifferentiated) IECs; however, T84 cells do not require Fak and HCT116 cells do not require PI3‐K/Akt‐1, in contrast to the other cells studied; (4) Src acts as a cornerstone in Fak‐mediated signaling to MEK/Erk and PI3‐K/Akt‐1 in T84 cells, as in undifferentiated IECs, whereas PI3‐K/Akt‐1 is Src‐independent in HCT116, HT29 cells, as in differentiated IECs; and (5) EGFR activity inhibition abrogates anoikis resistance in cancer cells through a loss of Fak–Src interactions and down‐activation of Src/MEK/Erk (T84, HCT116, HT29 cells) and PI3‐K/Akt‐1 (T84 cells). Hence, despite distinctions in signaling behavior not necessarily related to undifferentiated or differentiated IECs, intestinal epithelial cancer cells commonly display an EGFR‐mediated sustained activation of Src under anoikis conditions. Furthermore, such sustained Src activation confers anoikis resistance at least in part through a consequent sustenance of Fak–Src interactions and MEK/Erk activation, thus not only overriding Fak‐mediated signaling to MEK/Erk and/or PI3‐K/Akt‐1, but also the requirement of Fak and/or PI3‐K/Akt‐1 for survival. J. Cell. Biochem. 107: 639–654, 2009. © 2009 Wiley‐Liss, Inc.